Abstracts

INTRODUCTION

The World Health Organization (WHO) has established various strategies for controlling the Aedes aegypti, population, especially in the use of chemical and biological products integrated with environmental management programs capable of eliminating the larval forms and adult insects.³¹31  World Health Organization. Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Geneva; 1970. (Technical Report Series, 443).

Conventional chemical insecticides used to control Aedes aegypti have encouraged the selection of resistant populations. Increasingly strong doses are needed, leading to toxic effects when accumulated in human and animal tissue, and to environmental contamination.⁴4  Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saude. 2007;16(4):295-302. DOI:10.5123/S1679-49742007000400007^,3030  Viegas Júnior C. Terpenos com atividade inseticida: uma alternativa para o controle químico de insetos. Quim Nova. 2003;26(3):390-400. DOI:10.1590/S0100-40422003000300017
https://doi.org/10.1590/S0100-4042200300... Ongoing use of biological control using the Bacillus thuringiensis, israelenses (BTI) variety also encourages the selection of resistant A. aegypti populations.²²22  Paris M, Tetreau G, Laurent F, Lelu M, Despres L, David JP. Persistence of Bacillus thuringiensis israelensis (Bti) in the environment induces resistance to multiple Bti toxins in mosquitoes. Pest Manag Sci. 2011;67(1):122-8. DOI:10.1002/ps.2046

Plant-based compounds are the main source of new molecules with the potential to be inserted into biological systems.¹³13  Macías FA, Oliveros-Bastidas A, Marín D, Carrera C, Chinchilla N, Molinillo JMG. Plant biocommunicators: their phytotoxicity, degradation studies and potential use as herbicide models. Phytochemistry Rev. 2008;7(1):179-94. DOI:10.1007/s11101-007-9062-4 Natural insecticides meet the needs for alternatives to controlling resistant populations of Aedes aegypti, a vector for a variety of viruses. They can affect different stages of development through a variety of mechanisms.¹⁸18  Navarro-Silva MA, Marques FA, Duque L JE. Review of semiochemicals that mediate the oviposition of mosquitoes: a possible sustainable tool for the control and monitoring of Culicidae. Rev Bras Entomol. 2009;53(1):1-6. DOI:10.1590/S0085-56262009000100002

Azadirachta indica and the Carapa guianensis are from the Meliaceae family. There are various compounds that have a larvicidal action on Aedes aegypti, A. albopictus and Culex,²⁵25  Rossi JCN, Prophiro JS, Pedroso MF, Torquato MF, Emerick TV, Mendes S, et al. Uso do óleo de andiroba (Carapa guianensis - Meliaceae) como larvicida de Aedes aegypti (Diptera: Culicidae). Rev Soc Bras Med Trop. 2005;41:78.^,2727  Silva OS, Romão PRT, Blazius RD, Prohiro JS. The use of andiroba Carapa guianensis as larvicide against Aedes albopictus. J Am Mosq Control Assoc. 2004;20(4):456-7. as well as acting as an insecticide, repellent, antifungal, antimicrobial, acaricide, antifeedant and growth regulator. They are effective at low concentrations and are, for mammals, of low toxicity.¹⁷17  Nakatani M, Abdelgaleil SAM, Saad MMG, Huang RC, Doe N, Iwagawa T. Phragmalin limonoids from Chukrasia tabularis. Phytochemistry. 2004;65(20):2833-41. DOI:10.1016/j.phytochem.2004.08.010^,1818  Navarro-Silva MA, Marques FA, Duque L JE. Review of semiochemicals that mediate the oviposition of mosquitoes: a possible sustainable tool for the control and monitoring of Culicidae. Rev Bras Entomol. 2009;53(1):1-6. DOI:10.1590/S0085-56262009000100002

Melaleuca alternifolia belongs to the Myrtaceae family and is used for its antimicrobial, antiviral, antifungal, antiseptic, anti-inflammatory and healing actions.¹⁰10  Hammer KA, Carson CF, Riley TV. Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. J. Antimicrob Chemother. 2004;53(6):1081-5. DOI:10.1093/jac/dkh243^,1515  Mondello F, De Bernardis F, Girolamo A, Cassone A, Salvatore G. In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species. BMC Infect Dis. 2006;6:158. DOI:10.1186/1471-2334-6-158 The oxygenated monoterpenes present in M. alternifolia essential oil are toxic to Aedes albopictus larvae and lethal concentration (LC₅₀) of 267.13 ppm.⁶6  Conti B, Flamini G, Cioni PL, Ceccarini L, Macchia M, Benelli G. Mosquitocidal essential oils: are they safe against non-target aquatic organisms? Parasitol Res. 2014;113(1):251-9. DOI:10.1007/s00436-013-3651-5

Carica papaya is from the Caricaceae and has bactericidal and bacteriostatic properties, and is used as a dewormer, facilitating digestion, reducing lipid peroxidation and an antioxidant.¹⁴14  Mello VJ, Gomes MT, Lemos FO, Delfino JL, Andrade SP, Lopes MT, et al. The gastric ulcer protective and healing role of cysteine proteinases from Carica candamarcensis. Phytomedicine. 2008;15(4):237-44. DOI:10.1016/j.phymed.2007.06.004 Fermented extract of C. Papaya leaf has larvicial, ovicidal and repellent actions against Aedes aegypti.⁹9  Govindarajan M. Bioefficacy of Cassia fistula Linn. (Leguminosae) leaf extract against chikungunya vector, Aedes aegypti (Diptera: Culicidae). Eur Rev Med Pharmacol Sci. 2009;13(2):99-103.

In isolation, all of the components of the compound possess larvicidal properties, although there have been no studies on their efficacy when blended to form one single product.

The aim of this study was to evaluate the efficacy of the compound of Azadirachta indica, Melaleuca alternifolia, Carapa guianensis essential oils and fermented extract of Carica papaya on Aedes aegypti larvae (Linnaeus, 1762) (Diptera: Culicidae).

METHODS

The essential oils and fermented extract compound is a commercial product obtained from Gued’s Biotecnologia^®. Its formulation is as follows: essential oil from Azadirachta indica seeds 10.0%, essential oil from Melaleuca alternifolia fruit 0.3%, essential oil from Carapa guianensis 1.0%, bacterial fermented extract of Carica papaya fruit 5.0%.

The essential oil and fermented extract compound is immiscible in water and forms a film on the surface of the container, causing the larvae to die of asphyxiation. It needed to be dissolved in an organic solvent, dimethyl sulfoxide, to enable it to be mixed with water. This was tested separately to analyze its toxicity for Aedes aegypti larvae.

Aedes aegypti Liverpool colonies were established from strains from the Universidade Federal Rural de Pernambuco, Laboratory of Domestic Animal Parasitic Diseases insectarium, Recife, PE, Northeastern Brazil, 2013. They were kept in a room with controlled temperature of 28°C (SD = 1), 80.0% (SD = 5.0) relative air humidity and a natural 12/12h photoperiod cycle.

Plastic containers holding two liters of de-chlorinated water were used to hatch the larvae. They were fed industrialized powdered cat food.

The toxicological trials followed the methodology recommended by the WHO.¹¹11  Jang YS, Kim MK, Ahn YS, Lee HS. Larvicidal activity of Brazilian plant against Aedes aegypti and Culex pipiens (Diptera: Culicidae). Agri Chem Biotechnol. 2002;45(3):131-4.^,3131  World Health Organization. Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Geneva; 1970. (Technical Report Series, 443). Three hundred larvae were collected and transferred to a disposable container holding 50 mL of de-chlorinated water (26°C to 28ºC) when they reached the third larval stage. Each test was conducted in triplicate, making 900 larvae in each experimental group, giving a total of 7,200 specimens. The larvae were exposed to the solutions for a 24h period and were monitored every 60 min. Larvae which survived the larvicide trial remained under observation until pupae and adult emerged. The behavioral parameters of the larvae were observed during the period of the experiment to verify alterations such as: stereotyped movement, forming clusters, agitation, lethargy, change of color, shedding exuviae and death.

The experimental groups were organized as followed: treated with A. indica, M. alternifolia and C. guianensis essential oils and bacterial fermented extract of C. papaya in concentrations of 50.0% (G1), 25.0% (G2) and 12.5% (G3) a positive control with Bacillus thuringiensis serotype israelensis (BTI) industrial larvicide at concentrations of LC₉₀ 0.37 ppm (PC1) and LC₅₀ 0.06 ppm (PC2) and a negative control with de-chlorinated water (NC1) and negative control with dimethyl sulfoxide (NC2).

The data concerning the compound’s efficacy were expressed using statistics describing centrality and dispersion trends (mean and standard deviation). The non-parametric Kruskal-Wallis and the Dunn post-hoc tests were used in order to analyze significance between the results and see which groups differed between themselves. The non-parametric Chi-square test (χ²) was used in the analyses regarding the behavior of the larvae during the 24h period. The GraphPad Software, Inc., 2000 program was used for analyses with a significance level of 0.05.

RESULTS

During the larvicide test, the behavior of the larvae in groups G1, G2 and G3 altered, p < 0.05, compared to that of those in NC1 and NC2 groups, 60 min after exposure to the compound. Movement gradually decreased, the larvae formed clusters and were lethargic, remaining immobile even when touched after three hours. The larvae in the positive control groups, PC1 and PC2, became lethargic two hours after exposure, remaining inert to touch and with dark, rigid cephalic capsule (Table).

The larvae in the negative control group NC1 and NC2 were fed and developed into pupae and adults within 72h of the experiment. However, the surviving larvae in G1 and G2 did not shed their exuviae and did not develop into pupae and adults during the 21 days following exposure. Compounds in concentrations of 50.0% and 25.0% inhibited their development.

Larvae in groups treated with the compound (G1, G2 and G3) had mortality rates of 65.0%, 50.0% and 78.0%, respectively, in the first ten hours of exposure, whereas the mortality rate in the positive control groups (PC1 and PC2) was 100%, p < 0.05, compared with NC1 and NC2. Larvae died in all of the treated groups. However, after 24h, the group with the most efficacious treatment was G3, in which 100% of the larvae died, comparable to groups PC1 and PC2.

Larvae in the negative control groups using water (NC1) and dimethyl sulfoxide (NC2) did not die in the 24h following exposure. The dimethyl sulfoxide used in diluting the compound did not provoke mortality in the NC2 group, indicating that it had no effect on larvae development or death in groups G1, G2 and G3.

A. aegypti larvae were susceptible to the compound of A. indica, M. alternifolia and C. guianensis essential oils and C. papaya fermented extract, especially at concentrations of 12.5%.

DISCUSSION

The compound of essential oils and bacterial fermented extract possessed hydro-soluble active substances with larvicidal properties on third stage Aedes aegypti Liverpool larvae. Such products, highly efficient, with low toxicity and little environmental contamination are preferred in studies on controlling culicidae larvae.⁵5  Caser CRS, Carlos GA, Gasperazzo W, Cruz ZMA, Silva AG. Atividade biológica das folhas secas de Neem, Azadirachta indica, sobre larvas de Aedes aegypti. Natureza on line [Internet]. 2007 [citado 2014 mar 28];5(1):19-24. Disponível em: http://www.naturezaonline.com.br/natureza/conteudo/pdf/03_CaserCRSetal_1924.pdf
http://www.naturezaonline.com.br/naturez... ^,2424  Resende MC, Gama RA. Persistência e eficácia do regulador de crescimento pyriproxyfen em condições de laboratório para Aedes aegypti. Rev Soc Bras Med Trop. 2006;39(1):72-5. DOI:10.1590/S0037-86822006000100014

The first sign of a product with larvicidal properties is decreased movement of the larvae.²⁶26  Ruiz LM, Segura C, Trujillo J, Orduz S. In vivo binding of the Cry11bB toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae). Mem Inst Oswaldo Cruz. 2004;99(1):73-9. DOI:10.1590/S0074-02762004000100013 Arruda et al³3  Arruda W, Oliveira GMC, Silva IG. Toxicidade do extrato etanólico de Magonia pubescens sobre larvas de Aedes aegypti. Rev Soc Bras Med Trop. 2003;36(1):17-25. DOI:10.1590/S0037-86822003000100004 showed how the movement of A. aegypti larvae decreased when treated with Magonia pubescens. Such a decrease was also observed in A.aegypti, Culex quinquefasciatus and Anopheles albimanus larvae when exposed to BTI.²⁶26  Ruiz LM, Segura C, Trujillo J, Orduz S. In vivo binding of the Cry11bB toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae). Mem Inst Oswaldo Cruz. 2004;99(1):73-9. DOI:10.1590/S0074-02762004000100013

The main active ingredient in A. indica essential oil is azadirachtin, which acts as a larvicide on A. Aegypti and is reported to cause irreversible physiological alterations.⁷7  Dua VK, Pandey AC, Raghavendra K, Gupta A, Sharma T, Dash AP. Larvicidal activity of neem oil (Azadirachta indica) formulation against mosquitoes. Malaria J. 2009;8:124. DOI:10.1186/1475-2875-8-124 Ndione et al¹⁹19  Ndione RD, Faye O, Ndiaye M, Dieye A, Afoutou JM. Toxic effects of neem products (Azadirachta indica A. Juss) on Aedes aegypti Linnaeus 1762 larvae. Afr J Biotechnol. 2007;6(24):2846-54. investigated the larvicidal action of A. Indica essential oil and found that 64.0% of fourth stage A. Aegypti larvae died at concentrations of 8 mg/L (1.0%), and 82.0% of larvae when the concentration was reduced to 3 mg/L (0.3%) in 24h exposure. This data showed the best performing larvicide in the G3, group treated with the lowest concentration of the compound.

The development of A. aegypti arvae exposed to A. indica was compromised. Azadirachtin blocks the synthesis and release of ecdysone,¹³13  Macías FA, Oliveros-Bastidas A, Marín D, Carrera C, Chinchilla N, Molinillo JMG. Plant biocommunicators: their phytotoxicity, degradation studies and potential use as herbicide models. Phytochemistry Rev. 2008;7(1):179-94. DOI:10.1007/s11101-007-9062-4 impedes shedding the exuvia and causes the cuticle to deteriorate,¹1  Aliero BL. Larvaecidal effects of aqueous extracts of Azadirachta indica (neem) on the larvae of Anopheles mosquito. Afr J Biotechnol. 2003;2(9):325-7. as well as blocking ecdysteroid protein receptors. This inhibits growth, and causes deformities, sterility and death in the larvae.¹⁶16  Murugan K, Hwang JS, Kovendan K, Kumar KP, Vasugi C, Kumar AN. Use of plant products and copepods for control of the dengue vector, Aedes aegypti. Hydrobiologia. 2011;666(1):331-8. DOI:10.1007/s10750-011-0629-0^,2929  Tateishi K, Kiuchi M, Takeda S. New cuticle formation and moult inhibition by RH- 5849 in the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae). Appl Entomol Zool. 1993;28(2):177-84.

Silva et al²⁸28  Silva OS, Prophiro JS, Nogared JC, Kanis L, Emerick S, Blazius RD, Romão PRT. Larvicidal effect of andiroba oil, Carapa guianensis (Meliaceae), against Aedes aegypti. J Am Mosq Control Assoc. 2006;22(4):699-701. studied the larvicide action of C. guianensis on all A. Aegypti Rockefeller larvae stages and reported that: LC₉₀ and LC₉₅ were 164 ppm and 182 ppm after 48h for first stage larvae; 212 ppm and 224 ppm for second stage; 210 ppm and 226 ppm for third stage; and 450 ppm and 490 ppm for fourth stage, respectively.⁸8  Emerick S, Prophiro J, Rossi J, et al. Resultados preliminares do efeito larvicida do óleo de andiroba (Carapa guianensis) (Meliacea) em mosquitos do gênero Culex (Diptera: Culicidae). Rev Soc Bras Med Trop. 2005;41:44-45. Third and fourth stage Aedes albopictus, Culex and A. aegypti larvae also died after using the oil from this plant at different dilutions.²⁵25  Rossi JCN, Prophiro JS, Pedroso MF, Torquato MF, Emerick TV, Mendes S, et al. Uso do óleo de andiroba (Carapa guianensis - Meliaceae) como larvicida de Aedes aegypti (Diptera: Culicidae). Rev Soc Bras Med Trop. 2005;41:78.^,2727  Silva OS, Romão PRT, Blazius RD, Prohiro JS. The use of andiroba Carapa guianensis as larvicide against Aedes albopictus. J Am Mosq Control Assoc. 2004;20(4):456-7.

There are various species of Melaleuca spp with larvicide actions against A. Aegypti, inclduing Melaleuca linariifolia, M. dissitiflora and M. quinquenervia, the essential oils of which obtained mortality of more than 80.0% in concentrations of 0.1 mg/mL in 48h of exposure.²¹21  Park HM, Kim J, Chang KS, Kim BS, Yang YJ, Kim GH, et al. Larvicidal activity of Myrtaceae essential oils and their components against Aedes aegypti, acute toxicity on Daphnia magna, and aqueous residue. J Med Entomol. 2011;48(2):405-10. DOI:10.1603/ME10108 However, in a study of larvicides conducted by Amer & Mehlhorn,²2  Amer A, Mehlhorn H. Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res. 2006;99(4):466-72. DOI:10.1007/s00436-006-0182-3M. quinquenervia oil in a 50 ppm solution caused mortality in 30.0% of third stage A. Aegypti larvae 24h after exposure.

Rawani et al²³23  Rawani A, Haldar KM, Ghosh A, Chandra G. Larvicidal activities of three plants against filarial vector Culex quinquefasciatus Say (Diptera: Culicidae). Parasitol Res. 2009;105(5):1411-7. DOI:10.1007/s00436-009-1573-z tested raw extract of Carica papaya, Murraya paniculata and Cleistanhus collinus on Culex quinquefasciatus larvae and observed the best larvicide activity in Carica papaya. This may be explained by the bioactive secondary metabolites in isolation or in combination. Kovendan¹²12  Kovendan K, Murugan K, Kumar AN, Vincent S, Hwang JS. Bioefficacy of larvicidal and pupicidal properties of Carica papaya (Caricaceae) leaf extract and bacterial insecticide, spinosad, against chikungunya vector, Aedes aegypti (Diptera: Culicidae). Parasitol Res. 2012;110(2):669-78. DOI:10.1007/s00436-011-2540-z tested raw extract of C. papaya leaf in isolation and obtained 92.0% mortality in A. aegypti larvae at a concentration of 500 ppm.

Controlling A. aegypti larvae and adults and Culex quinquefasciatus larvae using extract of C. papaya seed is due to inhibition of amylase, which reduces life span and fecundity in adults, as well as provoking mortality in larvae.²⁰20  Nunes NNS, Santana LA, Sampaio MU, Lemos FJA, Oliva ML. The component of Carica papaya seed toxic to A. aegypti and the identification of tegupain, the enzyme that generates it. Chemosphere. 2013;92(4):413-20. DOI:10.1016/j.chemosphere.2012.12.078^,2323  Rawani A, Haldar KM, Ghosh A, Chandra G. Larvicidal activities of three plants against filarial vector Culex quinquefasciatus Say (Diptera: Culicidae). Parasitol Res. 2009;105(5):1411-7. DOI:10.1007/s00436-009-1573-z

As in the above mentioned individual studies, in this article larvicidal activity remained even when associated with low concentrations of the essential oils and fermented extract, found in the commercial product; 1 mL contains 0.01 mg/L of A. indica, 0.003 mg/L of M. alternifolia, 0.01 mg/L of C. guianensis and 0.05 mg/L of C. papaya. This concentration is below those found used in isolation in the indexed journals, even when undiluted. Thus, the larvacidal efficacy remained.

To conclude, the mixture of A. indica, M. alternifolia, C. guianensis essential oils and C. papaya bacterial fermented extract act in synergy as a larvicide on Aedes aegypti, Liverpool at all concentrations in laboratory conditions. It is necessary to evaluate this compound against A. aegypti populations in the field and with larvae at other stages.

REFERENCES

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